Mineralization of bone and teeth is a complex process controlled by the interplay between various nucleators and inhibitors of hydroxyapatite crystal formation. Among these proteins, matrix Gla protein (MGP) is the most important with in vivo evidence for a role in regulating mineralization. Recently, we showed that MGP is induced by parathyroid hormone (PTH), and this response might play a crucial role in PTH-mediated inhibition of mineralization in osteoblasts. However, the transcriptional mechanisms involved in PTH regulation of MGP are not known. Detailed knowledge of this regulation will help in understanding a factor that can be used as a target for preventive and therapeutic interventions for patients with mineralization defects. The overall goal of this project is to use genetic and molecular approaches to determine the importance of MGP in the regulation of osteoblast mineralization by PTH; and to determine the transcriptional mechanisms involved in this regulation. This project will test two hypotheses: (a) induction of MGP is responsible for the inhibition of mineralization seen in osteoblasts following parathyroid hormone treatment, and (b) stimulation of MGP is mediated through activation of second messenger(s) which regulate specific cis-acting response elements in the promoter of the MGP gene. The following two specific aims will be pursued: 1. Determine the response of Mgp-deficient or -suppressed osteoblasts to parathyroid hormone treatment, and 2. Identify the DNA response elements necessary for MGP responsiveness to parathyroid hormone in osteoblasts. Aim 1 will use both Mgp-deficient (Mgp -/-) mice and osteoblasts in which MGP synthesis is suppressed using RNAi to determine the regulation of mineralization by PTH. We expect that PTH treatment of MGP-deficient/suppressed osteoblasts will not inhibit mineralization. Aim 2 will characterize the MGP promoter to understand the regulatory mechanisms involved in PTH induction. We expect, based on our preliminary data that CRE and AP-1 sites are key to PTH induced transcription of MGP. Our long-range goal, which will be tested in a future R01 application, is to characterize specific transcriptional factors involved in PTH-mediated regulation of MGP, and perform detailed structure-function analyses of MGP as it relates to PTH-mediated inhibition of mineralization.